Enhancement of differentiation of rat adipocyte precursor cells by pertussis toxin

To examine whether GTP-binding protein(s) is (are) involved in adipocyte differentiation, the effect of pertussis toxin (PT) was studied in rat adipocyte precursor cell culture. PT potentiated adipose conversion induced by dexamethasone, insulin, and 1-methyl-3-isobutylxanthine in a dose- and time-dependent fashion. Attenuation of an inhibitory control of adenylate cyclase was not the mechanism of action of PT. The dose-dependent inhibition of PT-catalyzed ADP-ribosylation of the Mr 40,000 protein of the cell membrane by preincubation of the toxin was inversely related to the potentiating effect on differentiation. PT-sensitive G protein(s) may be involved in adipocyte differentiation in a negative fashion.     

Catechol 2,3-oxygenase production by genetically engineered Escherichia coli and its application to catechol determination

Catechol 2,3-oxygenase was produced by Escherichia coli, harbouring the recombinant plasmid pBH100 which contained the pheB gene cloned from phenol-degrading Pseudomonas putida BH, and was applied for the determination of catechol in the liquor. E. coli JM103 (pBH100) and C600 (pBH100) showed, respectively, about 5 and 8.5 times higher activities than that of P. putida BH. Using the crude extract prepared from the culture broth of the recombinant, catechol between 0.1 and 3.0 g/ml could be determined quantitatively in phosphate buffer, synthetic sewage and in mixtures of phenol, benzoate and sallcylate, and also in sodium pyruvate solution. In addition to catechol, 3-methylcatechol, 4-methylcatechol and 4-chlorocatechol could be determined. Oxygenase activity of the crude extract was maintained completely during the 100-day storage at –20°C after being freeze-dried with 10% acelone.M. Fujita, M. Ike, Y. Kawagoshi and N. Shinohara are with the Department of Environmental Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565, Japan. T. Kamiya is with the Central Research Laboratory of Mitsubishi Electric Co., Amagasaki, Hyogo 661, Japan.     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.     

{beta}-Naphthyl oligophosphates: Inhibitors of photophosphorylation and H+-ATPase of spinach chloroplasts

ß-Naphthyl di-, tri- or tetraphosphate inhibits photophosphorylationof spinach chloroplasts competitively with ADP, whereas ß-naphthylmonophosphate inhibits it competitively with Pi. The apparentKi of ß-naphthyl diphosphate for the ADP site was300 µM and that of ß-naphthyl monophosphatefor the Pi site was 1.45 mM. At 10 mM, both of these two organicphosphates inhibited photophosphorylation more than 90%. Noneof the above four ß-naphthyl phosphates were phosphorylatedby chloroplasts. ß-Naphthyl di-, tri- or tetraphosphateinhibits ATPase activity of isolated chloroplast coupling factor1 (CF₁) (EC 3.6.1.3 [EC] ) and light-triggered ATPase activity ofchloroplasts competitively with ATP, whereas ß-naphthylmonophosphate acts non-competitively. None of the four ß-naphthylphosphates were hydrolyzed by these two ATPase activities. Atconcentrations equal to ADP or ATP, ß-naphthyl di-,tri- or tetraphosphate inhibited these three reactions in theorder; ATPase of isolated CF₁> photophosphorylation>light-triggeredATPase of chloroplasts. The results suggest that the effect of the monophosphate isprincipally on the Pi site(s) and that of the di-, tri- or tetraphosphateis on the adenine nucleotide site(s) on the active center ofCF₁. ¹Part of this work was reported at the 1979 Annual Meeting ofthe Japanese Society of Plant Physiologists (Nagoya, April 7,1979) and the 52nd Annual Meeting of the Japanese BiochemicalSociety (Tokyo, October 7, 1979). This work was supported inpart by Grants-in-Aid for Scientific Research from the Ministryof Education, Science and Culture, Japan (311808 and 311909). (Received November 14, 1979; )     

Age-dependent differences in the ratio of synthesis of delta-crystallin polypeptides in cultured vitreous-free lenses of chick embryos

We have shown previously that the synthesis of the lower molecular weight polypeptides of δ-crystallin is differentially reduced and the intracellular $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ ratio is markedly increased in the 15-day-old embryonic chick lens cultured for 3 hr without the vitreous body or in the presence of ouabain. Here we demonstrate that neither δ-crystallin synthesis nor cation concentration is affected in the cultured, vitreous-free 6-day-old embryonic chick lens unless it is treated with ouabain. These results show that the alteration in δ-crystallin synthesis promoted by removing the vitreous body of the embryonic cultured lens is a stage-specific phenomenon, and are consistent with our previous correlation between the ratio of synthesis of the δ-crystallin polypeptides and the intracellular concentration of electrolytes.     

Retinal photoreceptor neurons and pinealocytes accumulate mRNA for interphotoreceptor retinoid-binding protein (IRBP)

We have utilized cDNA probes and in situ hybridization techniques to define the subcellular localization of interphotoreceptor retinoid-binding protein (IRBP) mRNA in bovine and monkey retinas. Results suggest that the mRNA is mainly localized in rod photoreceptor neurons within the outer nuclear layer of the retina. IRBP mRNA is also abundant in cells of the pineal gland, strengthening the analogy between rod photoreceptor cells and pinealocytes.     

Purification and characterization of a novel esterase (beta-hydroxypalmitate methyl ester hydrolase) and prevention of the expression of virulence by Ralstonia solanacearum

AIMS: To screen novel micro-organisms and enzymes capable of degrading 3-hydroxypalmitic acid methyl ester (3-OH PAME), the quorum-sensing signal molecule (quormone), which regulates the virulence of Ralstonia solanacearum. METHODS AND RESULTS: Ideonella sp. 0-0013, a betaproteobacterium isolated from soil using the selective-enrichment culture method, was grown on plates containing 3-OH PAME as its main carbon source. beta-Hydroxypalmitate methyl ester hydrolase (betaHPMEH) purified from the supernatant of the Ideonella sp. 0-0013 culture exhibited high hydrolysing activity towards the ester bond of 3-OH PAME and eliminated the 3-OH PAME activity, thereby reducing the virulence of R. solanacearum. An Escherichia coli transformant of the betahpmeh gene expression vector degraded 3-OH PAME, and the crude enzyme from the transformant inhibited in vitro production of the R. solanacearum exopolysaccharide (EPS). CONCLUSIONS: The ability of betaHPMEH to hydrolyse 3-OH PAME inhibited the production of EPS by the R. solanacearum wild-type strain, indicating that betaHPMEH inhibits the effects of activation of virulence genes. This ability will be potentially useful for pest control of the wilt disease caused by this bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: This enzyme is the first protein that has been found to degrade a quormone other than N-acyl homoserine lactone.     

Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes

Although many effects of leptin are mediated through the central nervous system, leptin can regulate metabolism through a direct action on peripheral tissues, such as fat and liver. We show here that leptin, at physiological concentrations, acts through an intracellular signaling pathway similar to that activated by insulin in isolated primary rat hepatocytes. This pathway involves stimulation of phosphatidylinositol 3-kinase (PI3K) binding to insulin receptor substrate-1 and insulin receptor substrate-2, activation of PI3K and protein kinase B (AKT), and PI3K-dependent activation of cyclic nucleotide phosphodiesterase 3B, a cAMP-degrading enzyme. One important function of this signaling pathway is to reduce levels of cAMP, because leptin-mediated activation of both protein kinase B and phosphodiesterase 3B is most marked following elevation of cAMP by glucagon, and because leptin suppresses glucagon-induced cAMP elevation in a PI3K-dependent manner. There is little or no expression of the long form leptin receptor in primary rat hepatocytes, and these signaling events are probably mediated through the short forms of the leptin receptor. Thus, leptin, like insulin, induces an intracellular signaling pathway in hepatocytes that culminates in cAMP degradation and an antagonism of the actions of glucagon.